Gene therapeutic treatment of blood vessel associated disorders

ABSTRACT

A catheter containing an inflatable balloon, which is at its periphery provided with hollow extensions that communicate between the outside of the balloon and the lumen of the catheter for use in the gene therapeutic treatment of local disorders by transfer of a desired gene to a target cell or tissue being part of or being located in the vicinity of a blood vessel. The catheter is preferably the Infiltrator(R) catheter.

BACKGROUND OF THE INVENTION

The present invention relates to a catheter for use in gene therapeutictreatment of local disorders by transfer of a desired gene to a targetcell or tissue being part of or being located in the vicinity of a bloodvessel. The present invention in a first preferred embodiment relatesmore in particular to the local transfer of the nitric oxide synthase(Ms) gene into the wall of arteries injured by interventional proceduressuch as angioplasty or stenting. In another preferred embodiment theinvention relates to the gene therapeutical treatment of vasculatedtumors by transfer of genes encoding a product that may kill or inhibitgrowth of tumor cells and/or vascular cells.

Gene therapy as intended by the present invention involves the geneticengineering of cells of a subject in need or therapy. Throughgenetically engineering cells they will acquire one or more desirableproperties they did not or did no longer possess, for example theability to express a particular protein. As an alternative a cell may begenetically engineered to loose an unwanted property.

Conditions that can be treated by means of gene therapy through localdelivery of a gene of interest to a target cell or tissue are forexample restenosis and cancer.

Restenosis is a complex biological process, initiated by plateletadhesion and aggregation at the site of arterial injury Plateletactivation results in the release of a variety of vasoactive, growth,and mitogenic factors that stimulate vascular smooth muscle cell (VSMC)proliferation and migration, matrix formation, and the latefibroproliferative response. In addition, injury to the endothelialprotective barrier results in the loss of constitutively expressedendothelium-derived vasoactive factors including nitric oxide (NO),prostacyclin and bradykinin, which under normal circumstances play animportant role in vascular homeostasis.

Over the past 15 years, percutaneous transluminal coronary angioplasty(PTCA) has significantly altered the management of symptomatic coronaryartery disease. Despite its overall value in achieving immediatesymptomatic relief, restenosis occurs in 30 to 50% of patients within 3to 6 months. Restenosis following PTCA is caused by progressive elasticrecoil, extracellular matrix formation, and fibrointimal hyperplasia atthe site of angioplasty. However, in randomized clinical trials mostcurrently used pharmacological agents have failed to demonstrate anybeneficial effect on restenosis. The need therefore exists for a newform of treatment of this condition. Gene therapy is a very promisingprospect. Local transfer of genes encoding antiproliferative andangiogenic proteins has been effective in animal models of neointimaformation following angioplasty in peripheral arteries.

All established tumors, both primary and metastasized, that are largerthan a few millimeter in diameter are vascularized. In addition, distantmetastases usually emerge after migration of tumor cells from theprimary tumor through the blood or lymphatic circulation. Thus, allsolid tumors are in close contact with the circulation and, inprinciple, could be reached via the circulation. Gene therapeuticallyinfluencing tumor cells via the circulation is therefore very wellpossible. Moreover, killing of a solid tumor does not necessarily dependon gene transfer into the tumor cells themselves. Gene therapystrategies have been proposed where genetic material (e.g., the HSV-tkgene) is introduced into endothelial cells of the tumor vasculature(e.g., W096/21416). This should result in destruction of the tumorvasculature, ultimately leading to tumor necrosis.

Gene therapeutic treatment of conditions like restenosis and cancer thatare associated with blood or lymphatic vessels can thus be accomplishedvia the circulation.

However, the first important step in genetically engineering cells ingene therapy is being capable of efficiently transferring a vectorharboring the gene of interest to the cell.

Local adenoviral-mediated vascular gene transfer is currentlyaccomplished by different delivery devices, including double balloon,coated balloon and microporous balloon catheters, which by virtue oftheir design have only resulted in limited vascular gene transfer inanimal models. The double balloon catheter creates an isolated spacewithin the artery for instillation of vectors, but delivery/transductionefficacy is hampered by side branches within the central space of thelumen. A balloon catheter coated with a hydrophilic polymer containingplasmid DNA is currently used in a human gene therapy protocol forangiogenesis in peripheral arteries, but is less suited for coronarygene transfer because of washout after exposure to the blood stream.Microporous balloon catheters allow local high pressure delivery withjet-lesion formation including intimal disruption, medial dissection, orsubintimal haemorrhage but result in limited transgene expression in thecoronary vessel wall of large animals.

In view of the above problems encountered in local delivery of vectorsfor gene therapy to vessel walls or the vicinity of vessels with thehelp of catheters, it is the object of the present invention to providethe possibility of a more efficient local vector transfer system than iscurrently available, in particular for use in the treatment ofconditions and disorders associated with or occurring in the vicinity ofblood vessels, such as restenosis and solid tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cutaway view of a balloon and lumen catheter suitable foruse in the present invention;

FIG. 2 is a photomicrograph illustrating vascular cells post-therapy;

FIG. 3 is a photomicrograph illustrating the distribution of transgenicvascular cells post-therapy; and

FIG. 4 is a photomicrograph of a cross section of a control LAD artery.

BRIEF SUMMARY OF THE INVENTION

This object is achieved by means of using a catheter containing aninflatable balloon, which is at its perifery provided with hollowextensions that communicate between the outside of the balloon and thelumen of the catheter for use in the gene therapeutic treatment of localdisorders by transfer of a desired gene to a target cell or tissue beingpart of or being located in the vicinity of a blood vessel. Aparticularly suitable catheter is the Infiltrators® catheter.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment the catheter can be used for treatmentcomprises therapy or prophylaxis of restenosis by the local transfer ofthe nitric oxide synthase (NOS) gene into the wall of arteries injuredby interventional procedures such as angioplasty or stenting. In analternative embodiment the treatment comprises treatment of vasculatedtumors by transfer of genes encoding a product that may kill or inhibitgrowth of tumor cells and/or vascular cells.

In a preferred embodiment of the invention the delivery of the vector tothe target cell or tissue is accomplished by means of a catheter asdisclosed in European patent applications no. 753 322 and no. 768 098.In a particularly preferred embodiment the catheter is the so-calledInfiltrators® (InterVentional Technologies Inc., San Diego, Calif. USA),a multi-lumen balloon catheter allowing PTCA and simultaneous localintramural delivery of vectors. The Infiltrator® is a non-compliantangioplasty balloon (2.0-4.0 mm), covered by three injector plates, eachcontaining seven injector microports (height 0.254 mm, exit width 0.102mm) which are recessed in the balloon during introduction in the artery(FIG. 1). Once the balloon is inflated to 1 to 2 atmospheres, themicroports radially protrude through the internal plastic lamina (IEL)of the arterial wall. A separate lumen allows intramural vector deliverythrough all three channels over a short period of time (<15 seconds) viahand injection. The fluid infiltration (400 μl) via this catheter causesmild, localized, medial edema without intimal damage or separation ofthe vessel layers.

The recombinant vectors for use in the present invention are selectedfrom the following: retroviral vectors, adenoviral vectors,adeno-associated vectors, herpes vectors, plasmids. Of these retroviralvectors and adeno-associated vectors are preferred. The vectors may beincorporated in liposomes.

The treatment can be performed on any blood vessel, in particulararteries or veins that have been injured by interventional procedures,like angioplasty or stenting, As an alternative, the treatment may beperformed on vascularized (solid) tumors through delivery of the vectorsto the cells or tissues of the vascularized part of the tumor.

The gene of interest that is to be transferred to the target cell ortissue depends on the effect to be achieved.

In the case of injured arteries the gene is for example the nitric oxidesynthase (NOS) gene.

Genes affecting local cellular proliferation, migration and neointimalformation in the vessel wall can favorably affect the outcome of thestenotic process. In principal, gene products affecting vascular tonemay also influence the sequelae of injury to the vessel wall.

A variety of different genes encoding antiproliferative proteinsdelivered to cells within the vessel wall reduce neointima formation.They include proteins that act directly on smooth muscle cellproliferation such as cell cycle proteins, toxic gene products, orproducts of normal endothelium Alternatively, indirectly-acting geneproducts like vascular endothelial growth factor have been used. Thecellular targets of gene transfer have been both endothelium and smoothmuscle cells within the media and

The retinoblastoma (Rb) gene product functions as a cell cycle inhibitorto inhibit the proliferation of smooth muscle cells that characterizethe proliferative response. Adenovirus-mediated gene transfer of aconstitutively active, non-phosphorylatable form of Rb reduces smoothmuscle cell proliferation and restenosis following balloon angioplasty.

The p21 protein is an important negative regulator of cell cycleprogression in mammalian cells that functions by inhibiting cell cycledependent protein kinases and by binding to proliferating cell nuclearantigen, a DNA polymerase delta cofactor. Adenovirus encoding the p21protein reduces vascular smooth muscle cell proliferation and neointimaformation.

Similarly, gax is a homeobox gene normally expressed by vascular smoothmuscle cells that is downregulated following vascular injury to promotethe wound-healing response in the vessel wall. In balloon-injured rabbitiliac arteries, adenovirus-mediated gax overexpression in smooth musclecells prevents neointimal hyperplasia and luminal stenosis, but does notaffect re-endothelialization and endothelium-dependent vasomotion.

The herpes simplex virus Thymidine kinase (tk) gene functions as aselectively toxic gene product when combined with ganciclovir treatmentand kills transduced cells, hereby depicting the population ofproliferating cells within the target tissue. Local adenovirus-mediatedgene transfer of tk has been demonstrated to reduce neointima formationby 50% in injured porcine arteries, and the BrdU labeling index by 65%and neointima formation by 50% in a rabbit hyperlipidemic injury model.

Vascular endothelial growth factor (vegf) is an endothelial mitogenwhich acts to promote re-endothelialization of the vessel wall, limitthe exposure of underlying smooth muscle cells to mitogenic stimuli, andthereby reduce the neointimal proliferation of smooth muscle cellsfollowing balloon injury of normal vessels. In a rabbit model ofatherosclerosis, local adenovirus-mediated vegf gene transfer has beenshown to increase the minimal luminal diameter and thus reduce thedegree of post-angioplasty stenosis. In another study, an adenovirusvector expressing the specific clotting inhibitor hirudin was used tolocally transduce smooth muscle cells and inhibit active thrombin in aninjured rat carotid model systemic effects on clotting were not observedas evidenced by unchanged partial thromboplastin times, but neointimaformation in the vessel was inhibited by 35%. The rationale underlyingthis study is that the inhibition of thrombin reduces the exposure ofthe injured vessel segment to cytokines anchor growth factors releasedlocally at the site of injury by activated monocytes, andothelial tells,or platelets within a blood clot.

The constitutive endothelial nitric oxide synthase (ceNOS) enzyme isresponsible for the local production of nitric oxide (NO). ceNOS genetransfer smooth muscle cells in rat carotid arteries denuded ofendothelium by balloon injury can also have profound local effects. Byincreasing cGMP levels and inhibiting smooth muscle cell proliferationat the site of injury, ceNOS gene transfer is an effective therapy toreduce neointima formation within the injured segment.

furthermore, antimigratory proteins can also be used to inhibit theprocess of smooth muscle cell migration that characterizes cellularremodeling of the vessel wall, The serpin plasminogen activatorinhibitor 1 (PAI-1), a specific inhibitor of both urinary plasminogenactivator (uPA) and tPA, is another attractive candidate protein totreat vascular stenosis based on the results of gene targetingexperiments in mice. The results obtained with vascular injury models inmice lacking genes encoding various components of the fibrinolyticsystem demonstrate that plasminogen activation is involved in theprocess of smooth muscle cell migration and vascular stenosis. Further,mice lacking urinary plasminogen activator (uPA) responsible forcellular plasminogen activation and degradation of extracelular matrixshow significant reductions in the vascular wound healing response thatleads to stenosis, while conversely, plasminogen activator inhibitory(PAI-1) deficient mice are hyperstenotic and show accelerated smoothmuscle cell migration and proliferation responses. PAI-1 gene transferinto PAI-1 knockout mice has been performed using adenovirus-mediatedgene transfer Into the liver to augment systemic expression of PAI-1.The results demonstrate that restoration of circulating PAI-1 into suchmice can suppress the vascular wound healing response and dramaticallyreduce the degree of vascular stenosis which develops following injury.Interestingly, no deleterious side effects on homeostasis by PAI-1overexpression have been observed in these mice.

In related studies on degradation of the extracellular matrix byvascular smooth muscle cell expression of metalloproteinases,adenovirus-mediated overexpression of the tissue inhibitor ofmetalloproteinase-2 (TIMP-2) was found to inhibit smooth muscle cellmigration through Matrigel in vitro, which can also be a useful strategyin vivo. Since plasminogen activation has been shown to activate matrixmetalloproteinases, this observation supports the hypothesis that uPA isa primary mediator of smooth muscle cell migration and neointimalthickening. Conversely, gene transfer in normal rabbits has been shownto increase neointimai thickening and augment contractile responses invein grafts transduced by a recombirant adenovirus vector expressingTGFB1-transduced vessels which increases smooth muscle cell migrationand proliferation in the vessel wall.

Genes whose expression will alter vascular tone can also havesignificant effects on blood vessel remodeling and other processes. Forexample, NO or endothelium-derived relaxing factor, has potentrelaxation properties on blood vessels. Instillation of adenovirus intothe lumen in normal rabbit vessels and endothelial cell gene transferusing virus encoding ceNOS results in augmented ceNOS synthesis,increases in basal CGMP levels, and both diminish contractile responsesto phenylephrine and enhanced relaxations to acetylcholine. Similarly,gene transfer of β2-adrenergic receptors was shown to augment expressionsix-fold and thus enhance the vasorelaxation induced by isoproterenol inde-endothelialized rat carotid arteries. Conversely, adenoviral genetransfer of the endothelin cDNA to the livers of rats led topathophysiologic levels of endothelin expression and systemichypertension mediated by the ETA receptor.

The effects described hereinabove are in part previously publishedobservations. However, they are indications that a broader scope ofprotection than gene therapeutic treatment with NOS as described in theExamples alone is indeed justified. NOS has been used as a model systemto show that the method of the invention is indeed very effective. Thisis not an effect that is limited to the NOS gene but applies to othergenes suitable for gene therapeutically treating restenosis as well.

The same applies to the treatment of cancer. The genes that can bebrought into a tumor cell or its vicinity may encode molecules that canbe used to kill tumor cells. Such molecules include but are notrestricted to suicide enzymes that convert a non-toxic prodrug into atoxic compound (e.g. the HSV-tk/ganciclovir system), cytokines,antisense nucleic acid molecules, ribozymes, and tumor suppressorproteins (such as, e.g. the Retinoblastoma or p53 gene products). Inaddition, treatment of cancer by gene therapy methods also includes thedelivery of replicating adenovectors that are toxic to the tumor cellsby themselves.

Gene therapy by introduction of nucleic acid molecules encoding suicideenzymes has been widely tested on a variety of tumor models. Especiallythe transfer of the Herpes simplex virus thymidine kinase (HSV-tk) geneinto tumor cells in conjunction with systemic administration of thenon-toxic substrate ganciclovir has proven to be an effective way ofkilling tumor cells in vivo. An important advantage of theHSV-tk/ganciclovir system is that upon ganciclovir treatment HSV-tktransduced tumor cells mediate a significant killing effect onneighboring untransduced tumor cells, the so-called bystander effect.Thus, using this approach there is no absolute need for gene transferinto every individual cell in a solid tumor to achieve successful genetherapy.

In an alternative embodiment the gene to be transferred can encode acytokine. Gene therapy for cancer by the introduction of nucleic acidmolecules encoding cytokines is based on the concept of enhancing theimmune response against the tumor cells. The ultimate goal of thisapproach is to obtain regression of the treated tumor and simultaneouslyinduce such a high degree of immunity that coexisting metastases arealso destroyed. Compared to direct administration of a cytokine proteinthe gene transfer approach as suggested by the present invention has theimportant advantage of high-level production of the cytokine at the siteof the tumor, while systemic concentrations of the cytokine remain low.This avoids any pleiotropic and toxic side effects associated with saidcytokine, Signs of (partially) successful cancer treatment have beendescribed for tumor cells expressing IL- 1, IL-2, IL-3, IL-4,interferon-gamma, interferon-alpha, TNF-alpha, IL-7, G-CSF, GM-CSF, andIL- 12, The present invention further enhances the efficacy of thesetreatments.

Killing of tumor cells can also be accomplished by introducing into saidtumor cells genetic information that comprises or encodes nucleic acidmolecules with a sequence complementary to that of a nucleic acidmolecule that needs to be expressed in said tumor cell. The introducednucleic acid molecule or copies made thereof in situ prevent thetranslation of said nucleic acid molecule that needs to be expressed insaid tumor cell into its encoded protein by specific base pairing. Thetumor cells will then die as a consequence of a shortage in said encodedprotein.

A similar effect can be achieved by introducing into said tumor cells anucleic acid molecule that encodes or is itself a so-called ribozyme ordeoxyribozyme. In this case said nucleic acid molecule or copies madethereof in situ are capable of specifically cleaving a nucleic acidmolecule that needs to be expressed in said tumor cells, Also in thiscase, the tumor cells will die as a consequence of a shortage in saidencoded protein.

Another similar effect can be achieved by introducing into said tumorcells a nucleic acid molecule that encodes or is itself a so-calleddecoy molecule, in this case said nucleic acid molecule or copies madethereof in situ (nucleic acid molecules or proteins) are capable ofspecifically binding to a protein molecule that needs to be functionallyexpressed in said tumor cells. Functional expression here means thatsaid protein molecule is capable of exerting its natural biologicalactivity, at least in kind but preferably also in amount. The specificbinding results in functional inactivation of said protein molecule thatneeds to be functionally expressed in said tumor cells. The tumor cellswill then die as a consequence of a shortage in functional expression ofsaid protein.

The present invention will be illustrated in the following examples thatare, however, not intended to be limiting the scope of the invention.

The present invention is illustrated in these Examples on the basis ofreduction of neointima formation and arterial restenosis followinginjury by gene transfer in the porcine coronary artery which is injuredby percutaneous transluminal angioplasty (PTA). The local delivery iscarried out with the Infiltrator® catheter which ensures efficient viraltransduction of medial smooth muscle cells (SMC) and of adventitialcells, resulting in increased local nitric oxide (NO) production andreduced smooth muscle cell proliferation and migration.

In addition, control studies were performed on the efficacy of theInfiltrator® for gene transfer into the vessel wall. These studiesdemonstrated that the Infiltrator® catheter enabled highly efficientlocal gene transfer in the media and adventitia of coronary arteries.

Furthermore, experiments were performed on the therapeutic effect ofadenovirus-mediated delivery of the cDNA encoding NOS, the enzymeresponsible for the formation of nitric oxide (NO) from L-arginine inintact blood vessels. NOS is either constitutively expressed inendothelium (ceNOS or type III), induced by cytokines in a variety ofcell types (iNOS or type II) or constitutively expressed in the brain(nNOS or type I). NO inhibits platelet aggregation, leukocyte adhesion,and VSMC proliferation and migration in vitro, and may be an importantendogenous inhibitor of vascular lesion formation in vivo. Oraladministration of L-arginine, the precursor of NO, is known to inhibitneointimal thickening 4 weeks after balloon denudation ofnormocholesterolemic rabbit iliac arteries. Recently, ceNOS genetransfer in the vessel wall, using the Sendai virus/liposome mediatedgene transfer technique, showed a 70% reduction of neointima formationin the rat carotid artery, but rats are notoriously sensitive topharmological and molecular modulation of neointima formation. In thepresent invention, a high efficacy of intracoronary ceNOS gene transferwas demonstrated and a significant reduction in arterial restenosis at28 days after balloon angioplasty in pigs, which respond to injury witha morphologic and pharmacosensitive pattern much more similar to that ofarterial restenosis in man.

The results of the Examples can be summarized as follows: (1) theInfiltrator® catheter allows a highly efficient localadenoviral-mediated transfer of genes, including NOS, into the medialand adventitial cell layers of non injured and balloon-injured coronaryarteries, (2) no transduction is observed in distal or unrelatedcoronary arteries, and (3) adenoviral-mediated transfer of cDNA encodingceNOS restores vascular CGMP levels following balloon injury andsignificantly reduces coronary restenosis 28 days after angioplasty,primarily via an effect on neointima formation.

In the Examples reference is made to the following figures:

FIG. 1. The Infiltrator® catheter incorporates a low pressurepositioning balloon 1 with three longitudinal strips 2 of sevenmicro-miniaturized injector ports 3 mounted on its surface. When thepositioning balloon is inflated, the injector microports radially extendand intrude a 15 mm vessel wall fragment at 120 ° angles.

FIG. 2. Photomicrograph illustrating the distribution of β-galactosidaseexpressing vascular cells following local AdCMV gal gene transfer in theLAD coronary artery. Recombinant, replication-deficient adenoviruscarrying a nuclear-localizing variant of the E. coli β-galactosidasecDNA (AdCMV gal, 300 μl, 5×10⁹ pfu/ml) was injected in porcine coronaryarteries using the Infiltrator® catheter. Marked transgene expression isobserved in medial SMCs and throughout the adventitia. The dark spotsrepresent the β-galactosidase.

FIG. 3. Photomicrograph illustrating the distribution of ceNOSexpressing vascular cells following AdCMvceNOS gene transfer in the LADcoronary artery as revealed by immunostaining for ceNOS. Markedtransgene expression is observed in the medial and adventitial cell.layers.

FIG. 4. Cross section of a control LAD artery from an uninfected animal,showing diffuse endogenous ceNOS immunoreactivity in luminal endothelialcells and in endothelial cells of vasa vasorum and a coronary sidebranch.

Examples 1 Construction and Purification of Recombinant Adenovirus

Recombinant adenovirus containing the human constitutive endothelialnitric oxide synthase (ceNOS) cDNA was constructed, and amplified aspreviously described (Jaxisens et al., J. Clin. Invest. (1996)98(2).317-324). Briefly, a 3.7-kilobaae fragment of cDNA encoding humanceNOS (Janssen et al., J. Biol. Chem. (1992) 267(3)) was cloned betweenthe enhancer/promoter of the cytomegalovirus (CMV) immediate early genesand the SV40 polyadenylation signal of the bacterial plasmid pACCMVpLpA(Gomez-Foix et al., J. Biol. Chem. 267:25129-25134 (1992)). The plasmidalso contains ElA-deleted sequences of type 5 adenovirus including theorigin of replication, the packaging signal and the pUC19 polylinker.

Recombinant adenovirus was generated through homologous recombinationwith PJMl7, a bacterial plasmid derived from the E3-deletion mutantdl309 (Bett et al., Virus Res. 39:75-82 (1995)), followingcotransfection in ElA-transformed human embryonic kidney (293) cells(Graham et al., J. Gen. Virol. 36:59-72 (1977)). The recombinant virusis both E1- and E3-deleted. ceNOS-containing viral isolates (AdCMVceNOS)were amplified on confluent 293 cells and, after appearance ofcytopathic effects, were isolated, precipitated, and concentrated bydiscontinuous CsCl gradient ultracentrifugation. For all in vivostudies, viral titers were adjusted to 5×10⁹ plaque forming units(pfu)/ml.

Recombinant adenovirus containing the inducible NOS (iNOS) cDNA wasconstructed using similar procedures. In addition recombinant adenoviruscontaining the LacZ gene encoding a nuclear-localizing variant of the E.coli β-galactosidase cDNA (AdCMVβgal (Herz & Gerard, PNAS 90:2812-2816(1993))), and similar constructs carrying the hirudin cDNA (AdCMvHir,construct made in the CTG by Dr P. Zoldhelyi), or no cDNA (AdRR5 (Kopleret al., Clin. Res. 41:211A (1993))) were used to evaluate gene transferefficiency, recombinant protein production or to serve as control virus.

Example 2

Adenovirus-mediated NOS Gene Transduction and Validation of RecombinantProtein Activity In Vitro

Porcine vascular smooth muscle cells (VSMCs) were cultured in DMEMsupplemented with l0o fetal bovine serum (Gibco BRL, N.V. LifeTechnologies, Merelbeke, Belgium), 50 units/ml penicillin and 50 g/mlstreptomycin. The cells were grown in chamber slides (Nunc Inc.,Naperville, Ill. USA) to approximately 60% confluence and infected withAdCMVceNOS or AdRR5 diluted in DMEM supplemented with 2% fetal bovineserum at 2, 20, and 200 pfu per cell. After 24 hours, the viralsuspension was removed, and the cells were maintained in culture for 3days.

The third day following infection, the cells were washed withphosphate-buffered saline (PBS), fixed for 20 minutes in 4%paraformaldehyde and washed twice in 1 mmol/L Tris, 0.9% NaCl, 0.1%Triton X 100 (Merck, Darmstadt, Germany), buffer pH 7.6 (Tris-bufferedsaline, TBS): Cells were preincubated with rabbit pre-immune serum(dilution 1.5) in TBS for 45 minutes and incubated overnight with amonoclonal mouse anti-human ceNOS antibody 2 μg/ml (1:125 dilution)(Transduction Laboratories, Exeter, UK), followed by incubation for 1hour with a rabbit anti-mouse IgG antibody complexed to horseradishperoxidase (dilution 1:200) (Vector Laboratories Inc., Burlingame, USA).Antibody binding was visualized with diaminobenzidine tetrahydrochloride(DAB) in 0.1 mol/L Tris buffer containing 0. 03% H₂O₂, pH=7.2. PBS wasused to wash the slides between incubation steps. Slides werecounterstained with Harris′ hematoxylin, dehydrated, and mounted withD.P.X. compound (Prosan, Gent. Belgium).

Cytoplasmic ceNOS immunoreactivity was observed in AdCMVceNOS-infectedVSMCs but not in AdRR5-infected VSMCs. The number of positive SMCs forceNOS-immunostaining was proportional to the multiplicity of infection(MOI) applied (45%±2% at MOI 2, 88%±4% at MOI 20, and 100%±1% at MOI200). Recombinant ceNOS expression after AdCMvceNOS infection Won alsoConfirmed by immunoblot analysis. Similar results were obtained withAdCMViNOS in cultured rat aortic SMCs.

ceNOS is an nicotinamide adenine dinucleotide phosphate (NADPH)- andtetrahydrobiopterin-dependent enzyme which catalyzes the oxidation ofthe terminal guanidino nitrogen of L-arginine to L-citrulline, withgeneration of NO. Electron transfer from reduced NADPH to nitrobluetetrazolium is accompanied by a colour change, which can be detected byhistochemical staining. Porcine VSMCs were infected for 24 hours withAdCMVceNOS or with a AdRR5 (2, 20 or 200 pfu/cell) as described above.The medium was changed 24 hours after infection, and cells wereharvested at 72 hours and incubated in 0.2 mmol/L nitroblue tetrazoliumwith 1 mmol/L NADPH in 0.1 mol/L Tris-HCl, pH7.2, and 0.2% Triton X-100for 30 minutes at 37° C. A dark blue cytoplasmic staining patternindicated NOS-dependent diaphorase activity and confirmed the presenceof functional enzyme. The number of cells expressing NADPH-diaphoraseactivity was similar to the number of ceNOS positive VSMCs.AdRR5-infected cells did not show NADPH-diaphorase activity at the MOI'sapplied.

Example 3 Gene Transfer Following PTCA in the Coronary Arteries in Pigs

All animal care and handling were performed in accordance with theguidelines specified in the National Institutes of Health “Guide forCare and Use of Laboratory Animals” and were approved by the Animal Careand Use Committee of the University of Leuven. Juvenile domestic pigsweighing 20-25 kg were treated with aspirin 300 mg orally, 5 days perweek, starting the day of surgery. They were anesthetized with anintravenous bolus of azaperone 0.1 ml/kg, pentobarbital 10 mg/kg andketa-mine 5 mg/kg, followed by an intravenous infusion of ketamine 10mg/kg/h. The pigs were intubated and ventilated with O₂ enriched-roomair. ECG and arterial pressure were monitored continuously throughoutthe experiment. After exposure of the right carotid artery, a 8F leftJudkins guiding catheter was introduced through a 8F sheath and advancedto the base of the aorta. The left main coronary ostium was then engagedand a commercial, non compliant 3.0 mm balloon dilatation catheter wasadvanced over a standard 0.014 inch flexible wire into the left anteriordescending (LAD) coronary artery, and positioned distal to the firstdiagonal branch, Arterial injury was achieved by three 30 secondsinflations at 10 atmospheres with a 1 minute reflow between eachinflation. The balloon was deflated and removed and the Infiltrator®catheter was advanced to the site of injury. The inflation of theInfiltrator® balloon at 2 atmospheres was followed by hand injectionover 10 seconds of 0.3 ml of recombinant virus (titer-5×10⁹ pfu/ml). TheInfiltrator® catheter was deflated, withdrawn and the right carotidartery ligated. Heparin was given during the experiment as a bolus of15,000 IU intra-arterially. After surgical repair of the neck cutdownthe animals were allowed to recover. Intra-muscular enrofloxacine, 0.5ml/10 kg, was administered for the first 3 days following theintervention. The animals were given normal chow and were reanesthetized4 weeks later for control coronary angiography. They were thensacrificed by saturated KCl injection and the coronary arteries wereharvested for morphometric analysis of the injured arterial segment asdescribed below.

Example 4 Adenovirus-Mediated Gene Transfer Efficiency In Vivo

To assess gene transfer efficiency of the Infiltrator® catheter in thecoronary vessel wall, 4 pigs were instrumented as described above, andwere infected with 300 μl AdCMVβal (titer 5×10⁹ pfu/ml) after balloonangioplasty. Intracoronary gene transfer was also performed in 2uninjured pigs. At day 4, the pigs were sacrificed, the heart quicklyremoved, and the LAD was cannulated, perfused with 4% formaldehyde under100 cm₂O . pressure for 4 hours, and washed with PBS for 24 hours. Toidentify the cells expressing the transgene, the artery was cut into2-mm rings, and cells expressing the LacZ gene were detected usingβ-galactosidase staining (3.3 mmol/L K₃Fe(CN) ₆, 3.3 mmol/L K₄Fe(CN), 1mmol/L MgCl₂ and 1 mg/ml5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, in Na phosphatebuffer, pH=7) for 8 hours at 37° C. The arterial rings were then washedin PBS and 5 μm paraffin sections at 0.2-mm intervals were prepared andcounterstained with nuclear fast red (Sigma, Bornem, Belgium) (FIG. 2).The number of medial and adventitial cells with blue coloration of theirnuclei was counted in the area of maximal transduction, and expressed asa ratio over the total number of cells.

In uninjured coronary arteries infected with AdCMVgal, markedβ-galactosidase activity was detected in medial and in adventitialcells, with occasional blue staining in the surrounding myocytes. Thepercent of β-galactosidase positive cells at the site of maximaltransduction was 41±10% in the media, and 23±3% in the adventitia (FIG.2). The β-galactosidase positive cells were mostly present in the outerlayers of the media and in the internal layers of the adventitia. Nonuclear blue staining was observed in the coronary arterial segmentsinfected with ADRR5 or AdCMVceNOS. The IEL was ruptured at the site ofpenetration of the injector microports without medial tear or rupture ofthe external elastic lamina (EEL). The number of β-galactosidasepositive cells was markedly increased around the site of the injectormicroports. In the arteries subjected to PTCA prior to viral infection,the injury was more pronounced with tear of the medial layers andintramural hematoma.

To quantitate recombinant protein production in vivo followingintracoronary gene transfer, porcine coronary arteries were transducedwith AdaMVHir (5×10⁹ pfu/ml). Secretion of recombinant hirudin inconditioned medium from explanted porcine coronary arterial segments wasmeasured by ELISA. Conditioned medium was changed daily from_3 to 7 andoccasionally up to 10 days after gene transfer. Briefly, the wells of ahigh-binding microtiter plate were incubated 48 hours at 4° C. with 1.0g/ml of a monoclonal anti-r-hirudin antibody obtained from Ciba-Geigy.Then, the coated plates were washed four times and blocked with 2tbovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing0.05% Tween 20 for 2 hours at room temperature. The plates were washedand conditioned medium and purified hirudin standards were applied tothe wells for 2 hours at room temperature. A horseradishperoxidase-conjugated sheep antibody recognizing r-hirudin (1:8000dilution in PBS) was added to the wells and incubated for 2 hours atroom temperature.

Antibody binding was visualized by a 15-minutes incubation at roomtemperature with orthophenylene diamine (OPD) in citrate buffer, pH 5.0.The reaction was stopped with 4 mol/L sulfuric acid and the absorbanceread at 492 nm. The detection limit of the ELISA (defined as lowestconcentration giving a signal above the mean+2SD of the background) was15 pg/ml. Recombinant hirudin production in conditioned medium fromAd-CMVhirudin-infected coronary arteries increased from 49 pg/ml at day3 to 200 pg/ml at day 7. After 7 days, the secretion decreased and wasno longer detectable at day 10. No hirudin secretion was detected inuntransduced right coronary arteries.

Example 5 Recombinant ceNOS Expression in the Vessel Wall FollowingIntramural Gene Delivery

Recombinant ceNOS expression in coronary arteries after AdCMVceNOSinfection was evaluated immunohistochemically on frozen arterialsections. Four pigs were infected with 300 μl AdCMvceNOS (titer 5×10⁹pu/tml) and sacrificed at day 4. Coronary arteries were harvested asdescribed.

5.1 ceNOS Immunostaining

Arterial rings were embedded in O.C.T. compound (Miles Inc., DiagnosticsDivision Elkhart, USA) and immediately frozen in liquid nitrogen.Coronary artery sections (5 m) were fixed for 20 minutes in ice-coldmethanol, washed, and incubated overnight with a monoclonal mouseanti-human ceNOS antibody 2 μg/ml (dilution 1:125) (TransductionLaboratories, UK) followed by incubation for 30 minutes in methanolcontaining 3% H₂O₂. The detailed experimental conditions are describedin example 2. Biotinylated rabbit anti-mouse antibody (dilution 1:200)was used as secondary antibody (Vector Laboratories Inc., Burlingame,USA) and antibody binding visualized by streptavidin-conjugatedhorseradish peroxidase (ABC kit, Dako). Diffuse ceNOS immunostaining wasobserved throughout the adventitia and in the outer cell layers of themedia (FIG. 3). In control arteries, only little ceNOS immunoreactivitywas detected in endot-helial cells of vasa vasorum, but no ceNOSimmunoreactivity was observed in medial SMCs (FIG. 4),

5.2 NADPH-diaphorase Staining

Four days after AdCMVceNOS infection 5 μm coronary artery cryosectionswere incubated with 0.2 mmol/L nitroblue tetrazolium and 1 mmol/L NADPHin 0.1 mol/L Tris-HCl, pH 7.2, and 0.2% Triton X-100 for 45 minutes at37° C. Uninjured, untranaduced coronary artery was used as control.Intense dark blue cytoplasmic staining, indicative of NADPH-diaphoraseactivity, was observed in the medial cells after infection. While thiswas seen in untransduced endothelium, NOS-dependent diaphorase activityWas not detected in the media of uninjured arteries or in injuredporcine arteries infected with AdRR5.

5.3 cGMP Measurements

No increases cGMP levels via stimulation of soluble guanylate cyclase.To test the ability of recombinant ceNOS protein to generate NO, cGMPproduction in the vessel wall was measured using a commercial enzymeimmunoassay (Amersham Life Science, Belgium). Frozen coronary arterialsegments from AdCMvceNOS-transduced, control injured and normal arteries(n=4, each) were homogenized in 1 ml ice-cold 6% trichloroacetic acid(TCA), pH 4.0, and centrifuged at 10,000 g for 15 minutes at 4° C. Thesupernatant was transferred into a 30-ml glass centrifuge tube and TCAwas extracted four times with H₂O-saturated ether. A 500 μl aliquot ofthe sample was then lyophilized, resuspended in 500 μl of 0.05 M sodiumacetate buffer, pH 5.8, and assayed for cGMP. Vascular cGMP levels wereexpressed as pmoles cGMP per mg of TCA-precipitable protein. VascularcGMP levels in untransduced injured arteries were significantly lowerthan in uninjured coronary arteries (0.33±0.20 vs 1.3±0.42 pmol/mgprotein, P=0.002). Adenoviral-mediated overexpression of ceNOS increasedvascular cGMP levels after injury to levels found in untransduced,uninjured arteries (1.8±0.98 pmol/mg protein).

Example 6 Effect of Local ceNOS Gene Delivery on Arterial Stenosis inPigs

In this study, 25 animals underwent local gene transfer with theInfiltrator® catheter following balloon angioplasty of the LAD coronaryartery. Intramural gene delivery was carried out with AdCMVceNOS (n=12)or AdRR5 (n=13). There were no significant differences in body weight,age, or gender between the two groups. (Table 1) The balloon to arterydiameter ratio was similar in both groups, excluding difference inballoon injury as a confounding variable.

TABLE 1. Group characteristics. AdCMVceNOS Characteristics AdRR5 (n =13) (n = 12) P value Body weight (kg) 26 3 25 2 ns sex  7/6 (F/M)  5/6(F/M) ns death 1 1 ns Balloon to artery 1.60 0.08 1.65 0.09 ns diameterratio occlusive thrombus 2 0 ns Pericarditis  2/13  1/12 ns Myocardialinfarction  2/13  0/12 ns Final analysis 10/13 10/12 ns

Twenty eight days after gene transfer, the pigs were sacrificed undergeneral anaesthesia by a lethal dose of saturated potassium chloridesolution. The coronary arteries were harvested, fixed with 4% formalinat 100 cm H₂O for three hours. The injured coronary segment was thencarrefully dissected from the epicardial surface and sectionedtransversely into 2 mm rings, washed, and embedded in paraffin. Five μmthick sections were cut every 100 μm and stained with Haemaluin andEosin (H&E) for subsequent analysis. Cross-sectional areas of the intimaand media were measured by an experienced observer blinded to the originof the samples, using a computarized morphometric analysis system (TCIImage, C. N. Rood N. V., Brufsel, Belgium; Media Cybernetics, SilverSpring, Maryland USA). The borders of the external elastic lamina (MeL),internal elastic lamina (IEL), and vessel lumen were traced on adigitizing board and the areas bounded by each were calculated. Thepercentage area stenosis (100×(1-lumen area/IEL, area), and the intima(I) to media (M) ratio (I/M ratio) were determined. The maximal(neo)intimal thickness (in mm) of each analyzed section was measured,and the extent of the injury was further assessed by the ratio of IELfracture length to IEL circumference. Animals were excluded if anocclusive thrombus was detected (in 2 pigs given AdRR5 and in none givenAdCMvceNOS). Random sections were reviewed by a second observer blindedto the treatment assignment.

Results are presented as mean ±SD. The paired Student's t-test was usedto compare percentage area stenosis, maximal intimal thickening,residual lumen area, and neointima area divided by IEL fracture lengthbetween the two groups. Differences were considered Significant atp<0.05.

H&E and Verhoeffs-Van-Gieson-stained sections from all arterial segmentswere examined. A small number of sections in AdRR5-infected arteriesshowed the presence of a hematoma in the media-adventitia dissectionplains as a result of the angioplasty, or occlusive thrombus. Nointramyocardial hemorrhage was observed.

Arteries transduced with AdRR5 showed a marked neointima at 28 days,consisting mostly of stellate and spindle-shaped cells in a looseextracellular matrix. In the majority of the samples, there was completecoverage of the luminal surface by a monolayer of endothelium-likecells. In AdCMVceNOS-treated animals, the neointimal area normalized tothe IEL fracture length and the maximal neointimal thickness weresignificantly smaller than in AdRR5-infected arteries (0.80±0.19 vs0.59±0.14 mm (p=0.011), and 1.04±0.25 vs 0.75±0.21 mm (p=0.012)respectively).

TABLE 2. Results of morphometric analysis of coronary artery segments at28 days after injury. AdRR5 AdCMVceNOS (n = 10) (n = 10) P value EELarea (mm²) 2.27 0.52 2.55 0.79 ns IEL fracture length/IEL (%) 0.40 0.100.43 0.13 ns Maximal Neointimal 1.04 0.25 0.75 0.21 0.012 Thickness (mm)Lumen area (mm²) 0.32 0.18 0.70 0.35 0.007 Neointimal area/IEL 0.80 0.190.59 0.14 0.011 fracture length (mm) Stenosis (%) 75 11 53 15 0.001

What is claimed is:
 1. A medical device comprising a catheter containingan inflatable balloon, which catheter is provided at its periphery withhollow extensions that communicate between the outside of the balloonand a lumen of the catheter for use in the gene therapeutic treatment oflocal disorders by transfer of a desired gene to a target cell or tissuebeing part of or being located in the vicinity of a blood vessel.
 2. Thecatheter as claimed in claim 1, wherein the treatment comprises therapyor prophylaxis of restenosis by the local transfer of the nitric oxidesynthase (NOS) gene into the wall of arteries injured by interventionalprocedures such as angioplasty or stenting.
 3. The catheter as claimedin claim 1, wherein the treatment comprises treatment of vasculatedtumors by transfer of genes encoding a product that may kill or inhibitgrowth of tumor cells and/or vascular cells.
 4. A method foradministering gene therapy comprising: 1) locating within a blood vesselin an area of an animal or patient to be treated a distal end of acatheter containing an inflatable balloon, said catheter being providedat its periphery with hollow extensions that communicate between theoutside of the balloon and a lumen of the catheter; and 2) passingthrough said lumen and said hollow extensions at least one gene therapyactive agent to deliver said agent to said area.
 5. The method accordingto claim 4, wherein said gene therapy active agent is nitric oxidesynthase (NOS) in an amount effective to treat injuries in vessel wallsdue to interventional procedures such as angioplasty or stenting.
 6. Themethod according to claim 4, wherein said gene therapy active agent isselected from the group consisting of a gene therapy anticancer agentand a gene therapy antivascular agent.